1. Conventional methods for Detecting bacteria in Urine
Urine originates as an ultrafiltrate of plasma and is normally thought to be free of bacteria as it moves from the upper urinary tract to the bladder. Therefore, bladder urine obtained by suprapubic needle aspiration, and perhaps urine properly obtained by a catheter inserted via the urethral meatus, should contain no bacteria. As it is voided from the body however, the urine is frequently contaminated with microorganisms which colonize the distal urethra and/or the perianal area. The most common non-invasive method of obtaining urine samples which attempts to minimize, but rarely completely eliminates contamination involves meticulous swabbing of the urethral meatus and periurethral areas with a bactericidal agents, followed by the collection of a mid-stream "clean-catch" specimen. By current teaching, the specimen should be examined within 1 hour of collection (two hours if the unpreserved urine specimen is refrigerated) to obviate proliferation of bacteria. Although most contaminating microorganisms are avoided by this "clean-catch" method, the collected samples may still contain some contaminants.
Quantification of the viable bacteria has been the essential parameter used for determining the presence of "significant" (i.e. clinically relevant) bacteriuria. Today it is generally accepted that "clean-catch" urines which on culture are shown to contain: (1) 10.sup.5 colony-forming-units per ml (cfu/ml) represent true urinary tract infection (i.e., "significant bacteriuria"); (2) from 10.sup.3 to 10.sup.5 cfu/ml represent probable infection (varying with the author); and (3) less than 10.sup.3 cfu/ml represent probable contamination. Although "significant" bacteriuria refers to at least the presence of 10.sup.5 cfu/ml of urine, consistent findings of 10.sup.4 -10.sup.5 cfu/ml probably represent more than mere colonization if the distal urethra, particularly if only a single species of organism is present.
Traditionally, direct microscopic examination of urine to detect and to approximate the number of bacteria in specimens has been performed according to four general methods. As described by C. Cobbs (in Urinary Tract Infection and Its Management, D. Kaye, ed., C. V. Mosby Company, St. Louis, Mo., ch. 4, pp. 43-44), these include: (1) A small sample of uncentrifuged urine is spread on a slide, covered with a cover slip, and examined with the high dry objective (at 400X magnification) i.e., a wet mount preparation). A dye such as methylene blue may be added to the urine to enhance visibility of the bacteria. (2) A small sample of uncentrifuged urine is placed on a slide, heat-fixed, stained, e.g., using Gram stain, and examined with the high dry objective (at 400X magnification) or under oil immersion (at 1000X magnification). (3) A known volume of urine is centrifuged, the sediment resuspended in the residual fluid. A small sample of the sediment is spread on a slide, covered with a cover slip, and examined directly using the high dry objective (i.e., a wet mount slide preparation). (4) The centrifuged urine sediment is prepared as in (3) above, but the sample is heat-fixed, stained, e.g., using Gram stain, and examined with the high dry or under oil immersion objective. Using the above methods, detection of one or more bacterial organisms per microscopic field has been correlated with the minimum bacterial counts obtained by conventional culture of urine samples according to Cobbs' data illustrated in Table I.
TABLE I ______________________________________ Bacteria Magnifi- Correlation with Urine Sample Observed Field cation Bacteria on Culture ______________________________________ Unstained 1 Organism 400 .times. 10.sup.6 per ml. Uncentrifuged or more Stained 1 Organism 1000 .times. 10.sup.5 per ml. Centrifuged or more Unstained 1 Organism 400 .times. 10.sup.5 per ml. Centrifuged or more Stained 1 Organism 1000 .times. 10.sup.4 per ml. Centrifuged or more ______________________________________
Under good conditions bacteria may be seen in an aqueous medium under the microscope at as low at 100 diameters or less magnification, but they are usually visualized at 1000 diameters magnification after drying and staining with appropriate dyes. In the past both methods of visualization have been used to examine urine for the presence of bacteria. Using conventional wet mount slide preparations, bacteria usually observed in urine are Gram negative bacilli (rods). Group D streptococci are also seen in classical urinary tract infections (Todd et al., 1984, in Clinical Diagnosis and Management by Laboratory Methods 17th ed., Henry, ed., W. B. Saunders, N.Y.).
Much more commonly bacteria are demonstrated in urine by allowing the bacteria to grow in a designated culture medium until the colonies are visible to the naked eye. By counting the colonies and multiplying by the dilution of the urine, and by assuming that one colony consists of the progeny of a single bacterium (or a small cluster of bacteria such as a pair) in the original specimen, the number of bacteria (or more accurately, colony forming units of bacteria) in a given volume of urine may be estimated.
Using conventional urine culture techniques, it has been uniformly reported since Kass in 1956 (Transactions of the Association of American Physicians, 69, 56-64) that the Gram negative bacillus E. coli. is the most common causative agent in acute urinary tract infections. In chronic urinary tract infections, especially those in which there are structural abnormalities (e.g., obstructive uropathy, congenital abnormalities, neurogenic bladder, fistulous communications involving the urinary tract etc.), infection is often associated with antimicrobial-resistant Gram negative bacilli such as E. coli, Proteus sp., Pseudomonas sp., and the Klebsiella-Enterobacter group. Finally, largely as a result of technology, urinary tract infections are associated with the presence of Gram negative rods such as E. coli, Proteus, the Klebsiella-Enterobacter group, and a lesser number of cocci such as Staphylococcus epidermidis, and the enterococcus.
In the literature since Kass in 1956, Gram positive organisms are much less frequent as causative agents than are Gram negative bacilli, and they are often observed as "contaminants" because they yield low colony counts. This skewing against the incidence of Gram positive organisms has been made worse by the selection of culture media for routing laboratory use. Since the incidence of Gram negative rods had been reported to be higher, media have been selected to favor the rapid growth of Gram negative rods at the expense of Gram positive cocci, and this selection of media has further distorted the incidence.
Direct microscopy and culture methods each have disadvantages. With regard to the direct examination of the urine, it must be noted that bacteria may be seen in urine at only 100 or more diameters magnification, but the size of the image is not the only consideration influencing visibility. Should the optical density and refractive index of an object be near that of the medium, then it would not be detected by an ordinary light microscopy. Such an object might be seen by appropriate staining with dyes or by specialized lighting such as dark-field illumination, phase illumination, or differential interference. Even then, as pointed out by Kunin (1961, New Eng. J. Med. 265: 589), round bacteria cannot be distinguished from other near round particles such as crystals. Moreover, in a staining procedure, if for any reason the bacterial preparation does not adhere to the slide, the preparation is lost.
In the past 20-25 years, direct visualization of bacteria in urine has largely been abandoned in favor of methods involving culturing and counting the colonies of bacteria. Indeed, virtually all of the studies of the significance of bacteriuria since 1956 are based upon culturing the urine, and direct microscopic examination of urine has been relegated to the status of a quick but inadequate screening procedure which may be helpful because it is rapid and can be correlated with the culture methods.
Any useful culture method requires that the bacteria will grow in the laboratory in the medium selected and in the time allotted. If the medium used is inappropriate for the growth of the particular organisms present, they will not grow. If the time allotted is too short, colonies will not be visible and positive cultures may be mistakenly reported as negative; and if oxygen tension or oxidation potential is either too high or too low, fastidious anaerobic or aerobic organisms may be missed.
If the bacteria are dead when excreted from the body, then they will not grow. Dead bacteria were once alive, and dead ones in the urine were probably alive in the body. They are unable to multiply in the bladder, an important consideration in Kass' rule of 10.sup.5 cfu/ml. In addition, there are many reasons why bacteria in urine might display less than optimal viability. For example, the ionic strength or osmolarity of the urine may be outside the requisite range. The wall of the bacterium may be damaged so that it will require a special medium to grow. The oxidation potential potential of urine may be too high for growth of a particular bacterial species (e.g., the typical oxidation potential of urine observed by me is about +0.22+0.25 Volts, referenced to a saturated calomel electrode. This potential is sufficiently high to inhibit the growth of many bacteria). There may be agents in urine which inhibit bacterial growth. For example, antibodies against bacteria have been identified in urine and they have been demonstrated to be deposited on bacteria in urine. Moreover, antibiotics administered to a patient may be excreted in urine in active form at a higher concentration than in other body fluids. The more concentrated antibiotic or antibiotic metabolite is likely to inhibit bacterial growth. Urea (and perhaps other metabolites) present in all urine inhibits bacterial growth. Any one or a combination of these factors may inhibit or diminish bacterial grown in vitro.
U.S. Pat. No. 4,225,669, issued on Sept. 30, 1980 to Melnick et al., describes methods for semi-quantitative and semi-qualitative detection of bacteria in fluid specimens including urine, blood, water, samples and pharmaceutical products. The methods employ compositions of chelating agents and basic dyes capable of staining bacteria at pH of 7 or greater and observing the stained bacteria with the naked eye. Neither culture nor microscopic examination of bacteria is necessary. By intention and design, Melnick's method is referenced to the cultures and is used as a screening procedure prior to culture.